The long-term goal of this project is to understand the etiopathogenesis of human gelastic epilepsy. Human hypothalamic hamartoma (HH) is a rare developmental malformation often characterized by gelastic seizures, which are refractory to medical therapy. Based on ictal EEG recordings from HH, the gelastic seizures are postulated to arise from the HH lesion itself. This idea is well supported by evidence of dramatic improvements in seizure control using a novel neurosurgical approach allowing for safe resection of HH. However, the mechanisms of epileptogenesis operative in this subcortical lesion are unknown. We have recently characterized, for the first time, the electrophysiological properties of single HH neurons acutely dissociated from surgical specimens. The objective of the proposed study is to explore a novel mechanism of epileptogenesis of human gelastic seizures using surgically resected HH tissues. The central hypothesis is that GABAA receptors expressed in HH neurons exhibit an excitatory phenotype, which may serve as a source of human gelastic seizures. The rationale for the project is that HH neurons may exhibit immature features due to or combined with their ectopic location and abnormal cytoarchitecture, as well as their persistent epileptic activity. This immaturity may underlie GABAergic excitation. The central hypothesis will be tested by pursuing two specific aims. Aim 1 is to characterize GABAA receptor-mediated excitation. The working hypothesis is that GABA exerts an excitatory role mediated through functional GABAA receptors. This idea will be tested by defining a dominant expression of functional GABAA receptors with a limited expression of functional ionotropic glutamate receptors, as well as showing a positive shift of the reversal potential of the GABAA receptor-mediated currents and a GABA-induced depolarization and excitation using gramicidin-perforated patch recordings in acutely dissociated HH neurons. Aim 2 is to determine levels of expression of the Na+-K+-Cl- cotransporter (NKCC1) and the K+-Cl- cotransporter (KCC2) in HH tissues. The working hypothesis is that HH tissues express an abnormally high ratio of NKCC1 to KCC2 genes, contributing to elevated intracellular CI- levels and excitatory, rather than inhibitory, GABA responses. Quantitative RT-PCR techniques will be used to measure NKCC1 and KCC2 mRNA levels. The planned studies will establish and test a novel hypothesis that enhances understanding of mechanisms involved in human gelastic seizures. Knowledge about excitatory GABAA receptor function of HH neurons will also aid selection of superior strategies for pharmaceutical development to prevent and control human gelastic seizures. [unreadable] [unreadable]